Universität zu Köln

Longarini, Edoardo José ORCID: 0000-0003-3919-1169 (2024). Mono-ADP-ribosylation Signaling in DNA Damage: New Tools and Biological Insights. PhD thesis, Universität zu Köln.

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Abstract

The repair of DNA damage is a key process to maintain the integrity of the genome and prevent dysregulation resulting in diseased states, such as cancer. To ensure efficient repair, this process is carried out by dedicated DNA repair pathways. The signaling cascade orchestrated to promote DNA repair relies not only on recruitment dynamics of specific repair factors, but also on the enzymatic synthesis of several post-translational modifications (PTMs). ADP-ribosylation (ADPr) is one of the most common PTMs in DNA repair, synthesized by the poly(ADP-ribosyl)polymerases PARP1 and PARP2. The biological and clinical relevance of this PTM is underscored by the therapeutic use of four FDA-approved PARP1/2 inhibitors against cancer. However, the chemical and structural complexity of ADPr resulted in a dearth of research tools, which severely limits our ability to study it. In this thesis, we address this key issue by the development of new research tools and methodologies for the study of ADPr. We then leverage these tools to investigate the regulation and functional outcome of this PTM in the DNA damage response. In the first study, we describe the development of a phospho-guided, chemoenzymatic strategy for the preparation of ADP-ribosylated peptides. We apply these peptides to develop a repertoire of new recombinant antibodies against several forms of ADPr, including site-specific and broad-specificity antibodies against mono-ADPr. We use these antibodies to discover that mono-ADPr is prevalent upon DNA damage, and describe its dependence on the hydrolases ARH3 and PARG. In the second study, we further improve the versatility and sensitivity of our antibodies by applying affinity maturation and the SpyTag/SpyCatcher technology. We leverage these reagents to discover that, in DNA damage, ADPr forms a two-phase signaling pathway characterized by a rapid but transient poly-ADPr signal followed by a delayed, long-lived mono-ADPr signal. We find that persistent mono-ADPr on histones recruits proteins to the site of DNA damage, including RNF114, an E3 ubiquitin ligase. We investigate the functional consequence of RNF114 to discover that it acts as a mono- ADPr effector protein to modulate the DNA damage response and telomere maintenance. Lastly, in an unpublished study, we turn our attention to aspartate- and glutamatelinked mono-ADPr. We find that, due to its high chemical lability, routine detection methods are unsuitable for the analysis of this PTM. This leads to a dramatic underestimation of its abundance in cellular processes. Here, we develop a method that reveals DNA damage-induced mono-ADPr on aspartate and glutamate residues by PARP1. Unexpectedly, we find that PARG, a poly-ADPr hydrolase that is thought to be inactive on mono-ADPr, can remove aspartate- and glutamate-linked mono-ADPr.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Longarini, Edoardo José elongarini@age.mpg.de orcid.org/0000-0003-3919-1169 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-721651
Date:	2024
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Außeruniversitäre Forschungseinrichtungen > MPI for Biology of Ageing
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language DNA damage, ADP-ribosylation, proteomics, PARP1, antibodies English
Date of oral exam:	25 January 2024
Referee:	Name Academic Title Matic, Ivan Dr. Schumacher, Björn Prof. Kraus, Lee W. Prof.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/72165